Hepatitis C virus (HCV) was first recognized in 1989. It infects the liver and is responsible for the majority of cases of non-A, non-B hepatitis (Alter and Seef, 1993). Infections are typically chronic and lifelong; many infected individuals are healthy and unaffected for decades, whereas others develop chronic hepatitis or liver cirrhosis, the latter often leading to hepatocellular carcinoma (Fry and Flint, 1997; Lauer and Walker, 2001). While screening of the blood supply has drastically reduced new transmissions of the virus, there exists a large cohort of infected individuals who will require treatment in the coming decades. Some reports estimate that nearly 3% of the world's population or about 170 million people worldwide, including about 4 million people in the U.S.A., are infected with HCV (Anon, 1999).
HCV infection and its clinical sequelae are the leading causes of liver transplantation in the U.S.A. No vaccine is currently available, and the two licensed therapies, interferon alpha and ribavirin, which are both non-specific anti-viral agents with incompletely understood mechanisms of action, are only modestly efficacious (McHutchison et al., 1998). Thus, whereas the best long-term response rates are obtained with a combination of interferon alpha-2b and ribavirin, only a minority of subjects treated with this combination achieves the desired result of no detectable serum HCV RNA six months after stopping treatment (McHutchison et al., 1998). Moreover, these drugs exhibit severe, life-threatening toxicities, including neutropenia, hemolytic anemia and severe depression. There is therefore an urgent need for the development of new therapeutic approaches and agents to combat HCV infection.
The development of new treatments for HCV infection would be facilitated by a detailed knowledge of how the virus attaches to and fuses with cell membranes, enters target cells, replicates therein, infects neighboring cells and induces disease symptoms. However, even a basic understanding of HCV replication and pathogenesis remains poor, primarily due to a lack of experimental models and key reagents. An important step, therefore, is the development of better model systems that will facilitate the elucidation of the mechanisms underlying various aspects of the viral life cycle and disease causation.
The HCV genome is a 9.6 kb positive-sense, single-stranded RNA molecule that replicates exclusively in the cytoplasm of infected cells (Rice, 1996). The genomic RNA encodes a ˜3000 amino acid polyprotein that is processed to generate at least ten proteins termed C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B (Grakoui et al., 1993; Rice, 1996; Lauer and Walker, 2001). The C protein constitutes the nucleocapsid; E1 and E2 are transmembrane envelope glycoproteins; p7 is a membrane spanning protein of unknown function; and the various non-structural (NS) proteins have replication functions (Bartenschlager and Lohmann, 2000; Op De Beeck et al., 2001).
The envelope glycoproteins are thought to play a crucial role in viral infectivity through their direct effect on various processes including the packaging of virions, the attachment of virions to target cells, fusion with and entry into these cells, and the budding of viruses from cell membranes before another round of cell infection can be initiated. HCV entry into target cells is a particularly attractive target for antiviral therapy because entry inhibitors do not need to cross the plasma membrane nor be modified intracellularly. In addition, viral entry is generally a rate-limiting step that is mediated by conserved structures on the viral envelope and cell membrane. Consequently, inhibitors of viral entry can provide potent and durable suppression of viral replication.
HCV entry into host cells requires attachment of the viral particle to the cell surface, followed by fusion of the viral envelope with the cellular membrane. The HCV envelope glycoproteins, E1 and E2, are thought to be involved in mediating virus entry into susceptible target cells. In mammalian cell-based expression systems, the molecular weight of mature, full length E1 is ˜35 kD and that of E2 is ˜72 kD (Grakoui et al., 1993; Matsuura et al., 1994; Spaete et al., 1992). E1 and E2 are present as a non-covalently associated heterodimer, hereinafter referred to as E1/E2, on the virus surface and undergo extensive posttranslational modification by N-linked glycosylation (Lauer and Walker, 2001).
Entry of the HCV structural proteins, C-E1-E2-p7, into the cell is followed by translocation into the endoplasmic reticulum (ER), which is accompanied by cleavage of internal signal sequences by ER-resident signal peptidases (Bartenschlager and Lohmann, 2000; Op De Beeck et al., 2001; Reed and Rice, 2000). It is assumed that HCV buds into the ER and matures by passage through cytoplasmic vesicles (Pettersson, 1991). Studies of the subcellular localization of HCV envelope glycoproteins and particles in cells transfected or infected in vitro suggest vesicle-based morphogenesis of HCV (Dash et al., 1997; Egger et al., 2002; Greive et al., 2002; Iocovacci et al., 1997; Pietschmann et al., 2002; Serafino et al., 1997; Shimizu et al., 1996). However, HCV-like particles have been detected in the cytoplasm of hepatocytes from infected patients, which suggests budding at the plasma membrane (DeVos et al., 2002), though the budding and maturation process of HCV have not yet been delineated.
The two most common experimental models of viral entry are cell-cell membrane fusion between receptor- and envelope glycoprotein-expressing cells, and entry of “reporter” viruses pseudotyped with heterologous envelope glycoproteins. Both systems rely on cell surface-associated expression of functional envelope glycoproteins. However, achieving expression of E1 and E2 on the surface of cells has proven to be elusive, and various studies have suggested that modification of the TM domains may be required. Two groups (Lagging et al., 1998; Takikawa et al., 2000) have described fusion and entry mediated by E1 and E2 ectodomains fused to the TM domain of the VSV G envelope glycoprotein. However, the TM domain of VSV G has no known dimerization function, and E1 and E2 were expressed from separate mRNAs, further minimizing their potential to form native heterodimers. It is unclear from these reports whether fusion and entry events were actually mediated by E1 and E2, because key controls demonstrating specificity were omitted.
There has also been some inconsistency in the results reported: one group showed that pH-independent entry of viral pseudotypes was mediated by either E1 or E2 (Lagging et al., 1998; Meyer et al., 2000; Lagging et al., 2002), whereas the other showed that pH-dependent fusion required both glycoproteins (Takikawa et al., 2000; Matsuura et al., 2001). Moreover, a more recent report that HCV-VSV chimeric envelope glycoproteins are not functional (Buonocore et al., 2002), contradicts the results of the earlier studies. It therefore appears that the chimeric VSV G system does not reproducibly model HCV envelope glycoprotein-mediated cell fusion and entry.
The apparent absence of E1/E2 heterodimers on the cell surface and the lack of N-glycan modifications by Golgi enzymes have led to suggestions that HCV envelope glycoproteins are retained in the ER (Duvet et al., 1998; Martire et al., 2001; Michalak et al., 1997; Patel et al., 2001; Selby et al., 1994). Both ER retention of E1/E2 and the heterodimerization of these glycoproteins are thought to be mediated by the TM domains of E1 and E2 (Cocquerel et al., 1999; Cocquerel et al., 1998; Cocquerel et al., 2000; Flint and McKeating, 1999; Flint et al., 1999; (Deleersnyder et al., 1997; Dubuisson et al., 1994; Op De Beeck et al., 2000; Patel et al., 1999; Ralston et al., 1993; Selby et al., 1994), and this has made it difficult to generate cell surface-expressed E1/E2 heterodimers. However, an experimental system for generating such surface-expressed E1/E2 heterodimers would be very valuable, with applications in, for example, the development of assays for measuring the extent of cell membrane fusion and pseudovirion entry and for identifying agents that inhibit HCV entry into susceptible cells, as well as the production of monoclonal antibodies and vaccines.